阅读说明：本技术 一种提高月季耐热性的基因RcHSP18.1及其应用 (Gene RcHSP18.1 for improving heat resistance of Chinese rose and application thereof ) 是由 明凤 李博文 李东世 张忍 郁昕雨 李玲敏 夏萌 于 2021-08-24 设计创作，主要内容包括：本申请公开了一种提高月季耐热性的基因RcHSP18.1及其应用,其特征在于,所述基因RcHSP18.1编码的蛋白序列如SEQ ID NO.3所示。本申请还公开了提高月季耐热性的方法,其特征在于,所述方法包括向所述月季引入基因基因RcHSP18.1,所述基因RcHSP18.1编码的蛋白序列如SEQ ID NO.3所示。(The application discloses a gene RcHSP18.1 for improving heat resistance of Chinese roses and application thereof, and is characterized in that a protein sequence coded by the gene RcHSP18.1 is shown in SEQ ID NO. 3. The application also discloses a method for improving the heat resistance of the Chinese rose, which is characterized by introducing a gene RcHSP18.1 into the Chinese rose, wherein the protein sequence coded by the gene RcHSP18.1 is shown in SEQ ID NO. 3.)

1. A gene RcHSP18.1 for improving heat resistance of Chinese roses, wherein the protein sequence coded by the gene RcHSP18.1 is shown as SEQ ID NO. 3.

2. The gene rchsp18.1 of claim 1, wherein the coding sequence of the gene rchsp18.1 is shown in SEQ ID No. 2.

3. The gene rchsp18.1 of claim 1, wherein the sequence of the gene rchsp18.1 is shown in SEQ ID No. 1.

4. The application of the gene RcHSP18.1 in improving the heat resistance of roses is characterized in that the protein sequence coded by the gene RcHSP18.1 is shown as SEQ ID NO. 3.

5. The gene RcHSP18.1 of claim 4 wherein the coding sequence of the gene RcHSP18.1 is shown in SEQ ID NO. 2.

6. The gene RcHSP18.1 of claim 4 having a sequence shown in SEQ ID NO. 1.

7. A method for improving heat resistance of a rose, which comprises introducing a gene RcHSP18.1 into the rose, wherein the protein sequence encoded by the gene RcHSP18.1 is shown in SEQ ID NO. 3.

8. The method for improving the heat resistance of roses according to claim 7, wherein the gene rchsp18.1 is located in an expression vector capable of expressing it in the roses.

9. The method for improving the heat resistance of roses according to claim 8, wherein the vector is pCAMBIA-2300.

10. The method for improving the heat resistance of roses according to claim 7, comprising:

(1) constructing a coding sequence of a gene RcHSP18.1 into an expression vector, wherein the coding sequence of the gene RcHSP18.1 is shown as SEQ ID NO. 2;

(2) transferring the expression vector containing the coding sequence of the gene RcHSP18.1 into China rose.

(3) Screening to obtain a Chinese rose plant containing the expression vector, wherein the expression vector contains the coding sequence of the gene RcHSP18.1.

Technical Field

The application relates to the field of plant breeding, in particular to a gene RcHSP18.1 for improving heat resistance of Chinese roses and application thereof.

Background

China rose (Rosa chinensis) is a Rosa plant of Rosaceae (Rosaceae), has a cultivation history of about 2000 years, has a wide cultivation range, and is often applied to urban greening, garden configuration and commercial production. China rose is one of ten famous flowers in China, and the first of the four cut flowers in the world enjoys the reputation of 'queen of flower', and is one of the most various famous flowers and trees in the world at present. The Chinese rose has beautiful flower shape, gorgeous flower color, strong flower fragrance, long flowering period and four-season flowering, and is widely popular in the market. China leads the tides in various fields such as China's rose gardens, production and cultivation, breeding and the like, flowers have become industries with great economic benefits at present, and the change of the quality of the flowers can improve the ornamental value and the economic value of the flowers.

China rose 'Anji La', born in Germany, belongs to the vine China rose. The branches and tendrils of the flower bud are soft or hard, the flower bud is luxuriant, the flower color is bright, the flower is small to medium, the flower clusters, small clusters are formed, the cup-shaped flowering mode is realized, the flowering period is longer, the continuous blooming in multiple seasons is realized, and the flower bud has high ornamental value and good disease resistance. In recent years, landscape construction is carried out in many regions by using 'Anji La' China roses, slope and wall greening is carried out in Shanghai mountain plantations by using 'Anji La' China roses from the initial stage of garden construction, and the landscape architecture is found to perform well even in the north of mountains. Based on the inspiration, the flower wall, the urban high frame and the overpass are built by utilizing 'Anjila' in the flower city construction of various cities such as Shanghai, Hangzhou and the like, the elevation landscape of the urban traffic channel is effectively improved, and the great demonstration effect is achieved.

High temperature is a major environmental factor that restricts growth and development of Chinese roses in summer: on the one hand, the high temperature in summer can cause the Chinese rose buds to stop differentiating, not bloom and lose ornamental value; on the other hand, the disease and insect pest of the Chinese rose are aggravated, and the growth and development of the Chinese rose are seriously influenced. Therefore, the research on the heat-resistant variety of the Chinese rose plays a crucial role in the development of the economic market of the Chinese rose. Therefore, those skilled in the art have made efforts to develop a gene for improving heat resistance of roses and use thereof.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the present application addresses the problem of improving the heat resistance of roses.

In order to achieve the above objects, the present application provides a gene rchsp18.1 for improving heat resistance of roses, wherein the protein sequence encoded by the gene rchsp18.1 is shown in SEQ ID No. 3.

In certain embodiments, the coding sequence of the gene rchsp18.1 is set forth in SEQ ID No. 2.

In certain embodiments, the sequence of the gene rchsp18.1 is set forth in SEQ ID No. 1.

On the other hand, the application also provides application of the gene RcHSP18.1 in improving the heat resistance of roses, and is characterized in that the protein sequence coded by the gene RcHSP18.1 is shown as SEQ ID NO. 3.

In certain embodiments, the coding sequence of the gene rchsp18.1 is set forth in SEQ ID No. 2.

In certain embodiments, the sequence of the gene rchsp18.1 is set forth in SEQ ID No. 1.

In another aspect, the present application also provides a method for improving heat resistance of a rose, comprising introducing a gene rchsp18.1 into the rose, wherein the protein sequence encoded by the gene rchsp18.1 is shown in SEQ ID No. 3.

In certain embodiments, the coding sequence of the gene rchsp18.1 is set forth in SEQ ID No. 2.

In certain embodiments, the sequence of the gene rchsp18.1 is set forth in SEQ ID No. 1.

In certain embodiments, the gene rchsp18.1 is located in an expression vector capable of causing its expression in the rose.

In certain embodiments, the vector is pCAMBIA-2300.

In certain embodiments, the method comprises:

(1) constructing a coding sequence of a gene RcHSP18.1 into an expression vector, wherein the coding sequence of the gene RcHSP18.1 is shown as SEQ ID NO. 2;

(2) transferring the expression vector containing the coding sequence of the gene RcHSP18.1 into China rose.

(3) Screening to obtain a Chinese rose plant containing the expression vector, wherein the expression vector contains the coding sequence of the gene RcHSP18.1.

The application analyzes the expression mode and the function of the China rose 'Angira' gene RcHSP18.1, shows that the expression mode and the function can improve the high-temperature resistance activity of prokaryotic escherichia coli DE3, can also improve the heat resistance activity of China rose plants by over-expressing the gene RcHSP18.1 in China rose, provides a convenient and efficient method for the research of the improvement of China rose heat-resistant varieties, and has important significance for widening germplasm resource innovation.

The conception, specific structure and technical effects of the present application will be further described in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present application.

Drawings

FIG. 1 shows the material used in this application, China rose 'Angira'.

FIG. 2 shows a diagram of prediction of the tertiary structure of the RcHSP18.1 protein in the present application.

FIG. 3 is a graph showing the results of expression analysis of the RcHSP18.1 gene in leaves of China rose 'Angira' treated at various temperatures (Normal temperature (NT)28 ℃ C., High Temperature (HT)50 ℃ C.) for 2 hours according to the present application. Where NT denotes a normal temperature of 28 ℃ and HT denotes a high temperature of 50 ℃.

FIG. 4 is a graph showing the results of expression analysis of the RcHSP18.1 gene in leaves of China rose 'Angira' at high temperature (50 ℃) for 0min, 30min, 60min, 90min, 120min, 150min, 180min, 210min and 240 min.

FIG. 5 shows growth curves of E.coli DE3 transformed with the RcHSP18.1 gene of the present application and E.coli DE3 of a control group at normal temperature (28 ℃). Wherein NT represents normal temperature, pET28a-HSP18.1 represents a recombinant vector pET28a strain transformed with RcHSP18.1 gene, and pET28a-EV represents pET28a no-load plasmid strain.

FIG. 6 shows growth curves of E.coli DE3 transformed with the RcHSP18.1 gene of the present application and E.coli DE3 of a control group at high temperature (50 ℃). Wherein HT represents high temperature, pET28a-HSP18.1 represents a recombinant vector pET28a strain for transforming RcHSP18.1 gene, and pET28a-EV represents pET28a no-load plasmid strain.

FIG. 7 is a graph showing the results of relative conductivity analysis of calli of China rose 'Angira' transformed with an overexpression vector of the RcHSP18.1 gene and control China rose 'Angira' after 2 hours of treatment at different temperatures (Normal temperature (NT)28 ℃ C., High Temperature (HT)50 ℃ C.) in the present application. Wherein AJL represents China rose 'Angira', WT represents a wild-type control group, and OX represents an RcHSP18.1 gene overexpression group.

FIG. 8 is a graph showing the results of gene expression analysis of calli of roses 'Angela' transformed with the RcHSP18.1 gene overexpression vector and control roses 'Angela' after 2h of treatment at different temperatures (normal temperature (NT)28 ℃ C., High Temperature (HT)50 ℃ C.) in this application. Wherein WT represents a wild-type control group, and OX represents an over-expression group of the RcHSP18.1 gene.

Detailed Description

The present application will now be further described with reference to examples, which are intended to be illustrative only, and the present application may be embodied in many different forms of embodiments and should not be construed as limited to the embodiments set forth herein.

Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as molecular cloning in Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. The reagents used are commercially available or publicly available reagents unless otherwise specified.

In the present application, various vectors known in the art, such as commercially available vectors including plasmids and the like, pET28a vector (available from Chongqing Youbao biotech Co., Ltd., product No. VT1207), pCAMBIA-2300 (available from Chongqing Youbao biotech Co., Ltd., product No. VT1383), Escherichia coli DE3 (available from Chongqing Youbao biotech Co., Ltd., product No. ST1007), and Escherichia coli DH5 α (available from Shanghai Bingo Biotech Co., Ltd., product No. TSV-A07) can be used.

The China rose variety Angirla (Angela) is a boneset China rose of Fenghua in 1984 of France, with pink flowers, bright spots in light pink colors and light center colors. No fragrance or mild fruit fragrance. 35 petals, the average diameter of the flower is 4 cm, the flower is small to medium, the flower is full-double (26-40 petals), the flower is clustered, small clusters are formed, the flower is in a cup-shaped flowering form, the flowering period is long, and the flower can bloom continuously in multiple seasons; the branches are medium and dense; leaves were medium, shiny, medium green. The height is 80-150 cm, and the longest length can reach 300 cm; widely planted on the elevated frame; good drought resistance, heat resistance and pollution resistance. The Chinese rose variety 'Angira (Angela)' of the application is purchased from Shanghai Bailin landscaping engineering Co.

Example 1 cloning of the China rose 'Angira' RcHSP18.1 Gene

Total RNA from leaves of wild type China rose 'Angela' (FIG. 1) was extracted using a SteadyPure Plant RNA Extraction Kit (commercially available), and total RNA was reverse-transcribed into cDNA using a reverse transcription Kit (commercially available). Using the reverse transcribed cDNA as a template, a coding region band of 465bp in length was obtained by PCR using the primer sequences shown in SEQ ID NO.4 (forward primer: 5'ATGTCGCTCATCCCAAATTTCC 3') and SEQ ID NO.5 (reverse primer: 5'TTACCCGGAAATTTCAATGGC 3'). The nucleotide sequence is shown as SEQ ID NO.2, the amino acid sequence coded by the nucleotide sequence is shown as SEQ ID NO.3, the nucleotide sequence consists of 154 amino acid residues, and the molecular weight is 19.7 kilodaltons. The full-length sequence of the RcHSP18.1 gene is shown in SEQ ID NO. 1.

Example 2 Chinese rose 'Angira' RcHSP18.1 protein bioinformatics analysis

(1) Selecting amino acid sequences of different species, analyzing the phylogenetic relationship of the RcHSP18.1 by using MEGA7, wherein the RcHSP18.1 and the Orchidaceae plant dendrobium officinale, Shenzhen pseudo-orchid are gathered in the same branch and belong to a monocotyledon branch.

(2) The tertiary structure of the RcHSP18.1 protein is predicted by https:// swissmodel.expasy.org/interactive, as shown in FIG. 2, the tertiary structure of the RcHSP18.1 protein is predicted to form a homodimer to play a role.

Example 3 analysis of expression patterns of the China rose 'Angira' RcHSP18.1 Gene after different temperature treatments

Respectively extracting total RNA in the leaves of China rose 'Angira' treated at different temperatures (normal temperature (28 ℃) and high temperature (50 ℃), reversely transcribing the total RNA into cDNA by utilizing a reverse transcription kit, and carrying out q PCR detection, wherein the method specifically comprises the following steps:

(1) the Chinese rose 'Angira' cultured in natural environment and in good growth state is taken, and the stem section with leaves is treated for 2 hours at normal temperature (28 ℃) and high temperature (50 ℃).

(2) Extracting total RNA of the 'Angira' leaves treated at different temperatures;

(3) reverse transcription kit is utilized to reverse transcribe total RNA into cDNA, primers SEQ ID NO.6 and SEQ ID NO.7 are designed according to SEQ ID NO.1, and qPCR detection is carried out.

A forward primer: 5'GTCGCTCATCCCAAATTTCC 3' (SEQ ID NO.6)

Reverse primer: 5'TTCTCGCCAGGAAATTCAGG 3' (SEQ ID NO.7)

As shown in FIG. 3, the gene was hardly expressed at room temperature, but the expression level under high temperature stress was significantly higher than that at room temperature.

Example 4 analysis of expression levels of the ` Angira ` RcHSP18.1 Gene at different stages of hyperthermia

The well-grown stem segments with leaves of Chinese rose Angira are treated at high temperature of 50 ℃ for 4 h. The total RNA of each period is extracted every 30min, is reversely transcribed into cDNA, and is subjected to Real-time PCR detection by using primers SEQ ID NO.6 and SEQ ID NO.7, and the result is shown in FIG. 4, wherein the expression level of the RcHSP18.1 is increased rapidly firstly and then is decreased continuously along with the time extension of high-temperature stress.

Example 5 analysis of the Effect of the 'Angila' RcHSP18.1 Gene on the Heat-resistant Activity of prokaryotes

The expression vector pET28a is transferred into escherichia coli DE3, and the heat resistance of the escherichia coli is verified after protein expression is induced, wherein the steps are as follows:

(1) designing primers SEQ ID NO.8 and SEQ ID NO.9, cloning the RcHSP18.1 gene by using 'anghalar' cDNA, and constructing a pET28a-HSP18.1 expression vector;

a forward primer: 5'CAAATGGGTCGCGGATCCATGTCGCTCATCCCAAATTTCC 3' (SEQ ID NO.8)

Reverse primer: 5'TGCGGCCGCAAGCTTGTCGACCCCGGAAATTTCAATGGCTTTG3' (SEQ ID NO.9)

(2) Coli DE3 strain containing the vectors pET28a-EV and pET28a-HSP18.1, respectively, was constructed.

(4) Diluting the newly shaken bacterial liquid OD to 1.0, and measuring a growth curve at the normal temperature of 28 ℃;

(5) adding IPTG into fresh bacterial liquid with OD of 1 to the final concentration of 1mM/L, culturing at high temperature of 50 ℃, and measuring a growth curve.

As a result, as shown in FIGS. 5 and 6, after the China rose 'anghala' RcHSP18.1 gene was transferred into the prokaryotic E.coli DE3, the RcHSP18.1 gene did not affect the growth activity of E.coli at normal temperature (FIG. 5) but enhanced the heat-resistant activity of E.coli at high temperature (FIG. 6) compared to the control group into which the empty vector pET28a-EV was transferred.

Example 6 analysis of the Effect of the 'Angila' RcHSP18.1 Gene on the Heat-resistant Activity of roses

(1) Primers SEQ ID NO.10(5'GGATCCTCTAGAACTAGTCATGTCGCTCATCCCAAATTTCC 3') and SEQ ID NO.11(5'GTCAGAAGGCCTCCCGGGCCCCGGAAATTTCAATGGCTTTG 3') are designed, an 'Angila' cDNA is utilized to clone the RcHSP18.1 gene coding sequence, the obtained RcHSP18.1 gene coding sequence is constructed into a vector pCAMBIA-2300, and the pCAMBIA-2300-HSP18.1 overexpression vector is obtained.

(2) Adding pure water and a small amount of liquid detergent into fresh and tender Anji leaf blades, shaking at 100rpm for 30-45min by a shaking table, washing, treating with 75% alcohol for 45s in a super clean bench, treating with 12.5% sodium hypochlorite for 6-10min, washing with sterile water for 3-5 times, cutting into leaf blades with the width of 0.2cm, inoculating into an explant induced callus culture medium (B), and growing fresh and tender callus tissue for 2-3 weeks; b, culture medium formula: MS +30g/L glucose +3 mg/L2, 4-D +0.05mg/L KT +2.7g/L plant gel.

(3) Extracting expression vector plasmid (the concentration needs to reach 1 mug/mul), transferring the expression vector into fresh Chinese rose callus of 'Angelica' by using a gene gun, and then inoculating into a callus proliferation culture medium (C), wherein the formula of the culture medium is as follows: MS +30g/L glucose +2mg/L NAA +0.01 m.g/L6-BA +2.7g/L plant gel.

(4) Proliferating for 1-2 generations, and then transferring into a screening culture medium (S) for screening to obtain transgenic callus; the formula of the S culture medium is as follows: MS +30g/L glucose +20mg/L hygromycin +2mg/L NAA +0.01 m.g/L6-BA +2.7g/L plant gel.

(5) In a tissue culture room at the temperature of 28 ℃, the Chinese rose 'Angira' callus is transferred into a proliferation culture medium for culturing for 20-30d after being screened by the screening culture medium.

(6) Taking wild type (callus not subjected to transgenic treatment) as a control, and respectively taking fresh callus of the wild type and the overexpression genotype to perform experiments;

(6) respectively processing at normal temperature (28 ℃) and high temperature (50 ℃) for 2 hours, taking part of callus materials to detect the relative conductivity value and the expression quantity of the gene RcHSP18.1.

(7) And detecting the relative conductivity value:

taking 0.2g of callus from each sample, and repeating for 3 times;

② adding 10ml of ddH₂O, vacuumizing;

standing for 30 min;

fourthly, detecting the conductivity value A of each sample;

boiling in boiling water bath for 20 min;

sixthly, detecting the conductivity value B of each sample;

(vii) relative conductivity (a B-1 100%

(8) Expression level of gene rchsp18.1:

extracting total RNA of two processed samples.

Reverse transcription kit is used to reverse transcribe total RNA into cDNA, and qPCR detection is performed with SEQ ID No.6 and SEQ ID No. 7.

Normal plant cells have cell membranes with selective permeability to substances, but when plants are in adverse conditions such as high temperature, drought, salt stress and the like, the permeability of the cell membranes is increased due to the damage of the cell membranes, so that electrolytes in the cells are leaked, and the measured conductance value is increased. According to the change of the difference value between the relative conductivity of the plants after the high-temperature stress and the relative conductivity of the plants under the normal condition, the stress resistance of different Chinese rose varieties under the high-temperature stress is weak. The results showed that the expression level of the RcHSP18.1 gene under high temperature conditions was enhanced after the overexpression vector containing the RcHSP18.1 gene was transferred into the callus of China rose 'Angelica' and treated at normal temperature or high temperature (FIG. 8); the overexpression of the rchsp18.1 gene significantly reduced the relative conductivity after high temperature treatment compared to the wild type (fig. 7), demonstrating that overexpression of the rchsp18.1 gene significantly enhanced the heat-resistant activity of rose 'anggila'.

The foregoing detailed description of the preferred embodiments of the present application. It should be understood that numerous modifications and variations can be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the concepts of the present application should be within the scope of protection defined by the claims.

Sequence listing

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